Showing posts with label experimental models. Show all posts
Showing posts with label experimental models. Show all posts

Tuesday, June 8, 2021

Wrap-up of 3rd International Blastocystis Conference

The 3rd International Blastocystis Conference took place Wednesday-Friday last week.

Organising committee:

  • Anastasios (Tasos) Tsaousis 
  • Eleni Gentekaki
  • Chr. Rune Stensvold
  • Funda Dogruman-Al

 Scientific committee:

  • Kevin S W Tan
  • Özgür Kurt
  • Eleni Gentekaki 
  • Chr. Rune Stensvold
  • Funda Dogruman-Al
  • Anastasios (Tasos) Tsaousis

 Sponsors included 

  • Microbiology Society
  • Biology - MDPI
  • PCR Biosystems

The conference was facilitated by Top Kinisis

The conference in numbers:

  •  3-day conference
  • 73 attendees
  • 29 countries represented
  • 66 abstracts
  • 38 oral presentations
  • 28 poster presentations
  • 265 tweets using #Blastocystis21 as hashtag
  • 3 awards

Awards:

Blastocystis Quiz Award: Mark van der Giezen (@MitoRem)

Poster Award: Adriana Marcela Higuera Gelvez (Draft genomes of Blastocystis isolates from human samples in Bogotá, Colombia)

Oral Presentation:

  • Winner: Jamie Newton (Investigating the metabolic fluctuations of the human gut during Blastocystis infection)
  • 1st runner-up: Lei Deng (Experimental colonization with Blastocystis ST4 is associated with protective immune response and modulation of gut microbiome in a DSS-induced colitis mouse model)
  • 2nd runner-up: Zuzana Lhotska and Vincent Billy (back-to-back presentations: Blastocystis colonization alters host immune response and gut microbiome, part a and b, respectively)

A special issue 'Putting Blastocystis on the Spot: Examining the Biology, Ecology and Epidemiology of a controversial gut microbe' has been launched in Frontiers. This Research Topic is linked to the conference. You can go here for more information.

The 4th International Blastocystis Conference is planned to take place in Chania, Crete, Greece in 2024.

Last, but not least a couple of screen capture impressions from the conference:



I will be back shortly with more updates from the conference.


R


Friday, October 19, 2018

2nd International Blastocystis Conference Wrap-Up - Part II

So, a lot of people would like to know about the take-home messages from the recent 2nd International Blastocystis Conference in Bogotá. There were many, and I might develop one more post to make room for more.

The first - and most important - thing I'd like to emphasise is that the community interested in Blastocystis is growing. And we're seeing a clearly multidisciplinary approach to studying the parasite. I think that this is what we need. The initial ideas about having Blastocystis-specific conference were developed by Funda Dogruman-Al and myself, and we both have a background in clinical microbiology. We have realised that in order to make sense of Blastocystis in a clinical microbiology (and infectious disease) context, we need research input from bordering fields, such as biology (genomics, cell biology, etc.), veterinary medicine (host specificity and impact of Blastocystis on animal health), gastroenterology (connection to microbiota and the extent of Blastocystis being involved in functional and inflammatory bowel diseases), bioinformatics (processing NGS data such as those pertaining to the profiling of gut microbiota communities), and ecology (people who are used to study interactions between organisms). At the conference, I believe that all (or at least most) of these fields were represented.

I was also thrilled to realise that many researchers have now adapted to the subtype terminology, - and even the allele terminology appears to be useful and pragmatic.


Status on the Blastocystis genome project. Slide by Andrew Roger.

Andrew Roger highlighted that the genomes of Blastocystis are more different than the genomes of human and mouse! Well-annotated genomes are available for ST1, ST4, and ST7, while draft genomes are available for subtypes 2, 3, 6, 8 and 9. 

 
What use are genomes? Summary provided by Andrew Roger.


Animal experimental modelling is possible. We know that rats can be colonised/infected by Blastocystis ST1 strain from a human and shed cysts in stool for more than one year.

Blastocystis is one of the few parasites that are really easy to culture and easy to get by. If we can learn to induce cysts in culture, these can be separated by sucrose gradient centrifugation or other methods and used for inoculation into volunteers, pigs, or rats, for instance. This can be used to study the impact of Blastocystis on the host, including immune system and gut microbiota. Baseline microbiota profiling is necessary prior to inoculation to know about the background variation in study individuals.

In terms of Blastocystis and gut microbiota: Since we published our conspicuous observations in 2015, many researchers have now corroborated our findings: Blastocystis is typically linked to increased microbiota richness and diversity; - something, which is generally considered a benefit and which is linked not only to gut health, but also to leanness. Especially the negative association between Blastocystis and Bacteroides has been highlighted by many now. It will be very interesting to learn why this is so. It also seems that Blastocystis are more common in individuals with a gut microbiota dominated by strictly anaerobes rather than facultative aerobes.

Faecal microbiota transplantaion (FMT): The recommendation of excluding FMT donors based on the finding of Blastocystis came up many times and was discussed in the context of the microbiota studies. It appear relevant to investigate further whether FMT donors should really be dismissed if they are Blastocystis-positive.

Some of the take home messages from Raul Tito Tadeo's talk.

In many animal groups, Blastocystis is a very common finding. These include mostly omnivores or herbivores. On the contrary, Blastocystis is very rare in strict carnivores, with no consistency in subtype distribution, indicating that these animals are not natural hosts of Blastocystis.The Blastocystis incidentially found in these hosts might stem from the prey that they have eaten.

Finally, I wish to highlight that there are excellent resources available from the pre-conference workshop, including an R script for microbiota analysis, and some tools for Blastocystis genome annotation. Please visit my previous blog post for links to these.

We cannot totally dismiss pathogenicity of Blastocystis; if existing, it may involve both strain- and host-specific factors.

And.... it's out: The time and venue for the 3rd International Blastocystis Conference will be Crete in 2021 (possibly June), with Eleni Gentekaki and Anastasios Tsaousis being involved in both the scientific and local organising committees... ! Please mark you calendars!

Andrew Roger, Raul Tito Tadeo, Kevin Tan and myself (taking the picture) enjoying some Club Colombia.


Tuesday, July 3, 2018

Experimental models for Blastocystis research - new paper out!

Experimental models are critical to advancing our knowledge on the role of Blastocystis in health and disease.

We have now published our work led by Dr Katerina Pomajbikova on the suitability of the rat as a model of Blastocystis colonisation. We observed that the rats were able to sustain the colonisation for more than one year, when a ST1 strain isolated from a human was used.

Next step could now be to monitor gut microbiota before and after challenge with Blastocystis cysts and look for changes at both individual and community level,, changes in alpha and beta diversity, etc.

The paper is free for download here until August 19, 2018.

Friday, August 30, 2013

This Month In Blastocystis Research (AUG 2013)

Quite a few papers relevant to Blastocystis research have made it to PubMed over the past month! Therefore, the August version of 'This Month in Blastocystis Research' is more like a list of papers + short descriptions/comments, rather than one or two actual paper reviews.

Dr Aldert Bart and his Dutch colleagues have published a study that confirms data emerging from other parts of Europe. Using microscopy (fixed faecal smears) and PCR, they found an almost 40% prevalence of Blastocystis in returning travelers with symptoms, and a prevalence of 18% in patients referred for other reasons. The distribution of subtypes found in the study population was quite similar to what has been found elsewhere in Europe with ST3 predominating (42%) and the rest of the subtypes attributable to ST1 (22%), ST2 (22%), ST4 (12%), ST6 (1%) and ST7 (1%).

The Tropical Parasitology theme issue on Blastocystis has now gone live. You’ll find a link to the editorial and the three papers included in the symposium here.

In my previous post I referred to a new study from Colombia which includes subtyping of Blastocystis isolates from humans, and a variety of animals, including birds. The paper is interesting for a number of reasons, but first and foremost it confirms the virtual absence of ST4 in humans in S America. Moreover, the study included 70 Blastocystis positive samples from asymptomatic carriers, 40 positive samples from patients with diarrhoea, and 15 positive samples form patients with IBS. Remarkably, all samples from healthy carriers were typed as ST1, those from patients with diarrhoea belonged to ST2, and those from IBS patients to ST3. Such a clear-cut distribution of subtypes across cohorts is unprecedented and of course warrants confirmation and further investigation. In Europe, ST4 is very common in humans, while it appears rare in humans in many other parts of the world. ST4 also appears rare among non-human primates (NHPs), our closest living relatives, and while NPHs and humans otherwise tend to share the same major subtypes (ST1, ST2, and ST3), this suggests that while subtypes 1, 2 and 3 have probably co-evolved with primates, ST4 has only recently entered the primate population with a preference for humans! I have hinted at this many times by now, but I find it extremely interesting which is why I keep repeating it.

There is a paper out by Santos and Rivera from the Philippines comparing microscopy of direct faecal smear with culture and PCR for detection of Blastocystis. They ended up concluding that culture was the best diagnostic modality, but it should be noted that the PCR used in the study targets a 1.8 kbp product (complete SSU rRNA gene!), and much smaller products are usually targeted in diagnostic PCR assays. The Blastocystis real-time PCR developed by me and my colleagues targets a sequence stretch of ~120 bp, securing optimum test sensitivity. The results of the Philippine study should be interpreted with this in mind.

Li et al., have published data on experimental infection of ST1 in Sprague-Dawley rats. Animals belonging to this species appeared susceptible to a ST1 strain isolated from a diarrhoeic patient that had been kept in culture and for which induction of cysts had been performed with a view to infecting the rats. The study confirms that Blastocystis is mainly a parasite of the coecum and colon. The authors found evidence of Blastocystis invasion into the lamina propria in one of the animals, and signs of inflammation in all animals challenged. While it is great to see that experimental models can be sustained and that encystation can be induced in vitro, at least two important factors must be kept in mind to fully comprehend the study: Although cysts were isolated by gradient centrifugation prior to inoculation, it is unlikely that all bacteria have been removed from cyst suspensions; in other words, the cyst preparation is not likely to be 'sterile', and any effect of the potentially accompanying bacterial flora is difficult to determine. Moreover, rats may not be natural hosts of ST1 (very few data available on the topic!), and so, the pathology caused in the rats may be an unlikely finding in humans, who are indeed natural hosts of ST1 and may have developed a high degree of tolerance to this subtype.

Are dogs, wolves, and other canids natural hosts of Blastocystis?

When visiting Australia earlier this month, I had the pleasure of meeting Wenqi Wang and Tawin Inpankaew, both PhD students working at School of Veterinary Science, The University of Queensland Gatton Campus and supervised by Dr Rebecca Traub. One of the foci of this group is to study Blastocystis in animals, for instance in households where animals are kept as pets. Recently, a paper emerged from this group on diversity of Blastocystis subtypes in dogs in different geographical settings, hence domestic/pound dogs from Brisbane, Australia, semi-domesticated dogs from a village in Cambodia, and stray dogs from Mumbai and other Indian cities. Using sensitive PCR methods they observed that almost one fourth of the Indian dogs were infected, while dogs in the Cambodian village and in Queensland remained largely uninfected. Coprophagy and access to Blastocystis-positive stool from different hosts may account for the relatively high prevalence in stray dogs in India, although one might assume that the prevalence would then be even much higher? The team used nested PCR in their study and found four different subtypes in the Indian dogs, including ST1, ST4, ST5 and ST6. Whether all of their data collectively indicate that dogs are not natural hosts of Blastocystis is a matter of debate and remains to be more thoroughly investigated. Indeed, prevalence and subtype data from studies of samples from wild life canids (dingos, jackals, wolves, coyotes, but also foxes and raccoon dogs) would shed further light on this topic.

Finally, for those interested in how Blastocystis deals with oxidative stress and related metabolic issues, there is a paper out on iron-sulphur cluster biogenesis in protozoan parasites by Ali and Nozaki citing works by Tsaousis (2012), Denoeud (2011), Long (2011), and Stechmann (2008).

Literature:

Ali V, & Nozaki T (2013). Iron-sulphur clusters, their biosynthesis, and biological functions in protozoan parasites. Advances in Parasitology, 83, 1-92 PMID: 23876871

Bart A, Wentink-Bonnema EM, Gilis H, Verhaar N, Wassenaar CJ, van Vugt M, Goorhuis A, van Gool T. Diagnosis and subtype analysis of Blastocystis sp. in patients in a hospital setting in the Netherlands. BMC Infectious Diseases, 13:289.

Li J, Deng T, Li X, Cao G, Li X, & Yan Y (2013). A rat model to study Blastocytis subtype 1 infections. Parasitology Research PMID: 23892480 DOI: 10.1007/s00436-013-3536-7

Parija SC (2013). Blastocystis: Status of its pathogenicity. Tropical Parasitology, 3 (1) PMID: 23961433

Parija SC, & Jeremiah S (2013). Blastocystis: Taxonomy, biology and virulence. Tropical Parasitology, 3 (1), 17-25 PMID: 23961437 

Ramírez JD, Sánchez LV, Bautista DC, Corredor AF, Flórez AC, & Stensvold CR (2013). Blastocystis subtypes detected in humans and animals from Colombia. Infection, Genetics and Evolution : Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases PMID: 23886615

Sekar U, & Shanthi M (2013). Blastocystis: Consensus of treatment and controversies. Tropical Parasitology, 3 (1), 35-9 PMID: 23961439

Stensvold CR (2013). Blastocystis: Genetic diversity and molecular methods for diagnosis and epidemiology. Tropical Parasitology, 3 (1), 26-34 PMID: 23961438  

Wang W, Cuttell L, Bielefeldt-Ohmann H, Inpankaew T, Owen H, & Traub RJ (2013). Diversity of Blastocystis subtypes in dogs in different geographical settings. Parasites & Vectors, 6 PMID: 23883734

Friday, June 29, 2012

On Blastocystis and Animal Models

I was recently encouraged by one of my readers to do a blog post on Blastocystis and animal experimental models. This is not exactly my core competence, which probably boils down to the fact that animal models have only been scarcely used in Blastocystis research for reasons that I will try to account for below.

Animal models (mice, rats, guinea pigs) have often been used to study interactions between hosts and microbes as well as the effect of chemotherapeutic interventions. Therefore, one might assume that animal models are an obvious way of potentially establishing a link between Blastocystis and pathology. But currently, the rationale for carrying out some types of Blastocystis experiments on, say, mice or rats is limited. Why? Well, first and foremost because of at least three major issues.

1) Lack of correlation between in vitro and in vivo evidence. Experimental infections of laboratory mice (Elwakil and Hewedi, 2010) resulted in tissue invasion - something never reported in humans. Another study showed increased oxidative stress in Blastocystis infected rats (Chandramathi et al., 2010), again something not linked to human colonisation. Studies that provided evidence for induction of cytokines, contact mediated apoptosis, and barrier disruption all used axenic Blastocystis and in vitro mammalian cell cultures with no evidence that these effect occur in vivo.

2) Host specificity. Blastocystis exhibits extreme genetic diversity and multiple, genetically very different variants (species, subtypes) exist. These subtypes exhibit moderate host specificity. This means that some subtypes are common in one type of host, whereas other subtypes are common in other types of hosts. For instance, ST5 is very common in pigs, but we rarely see it in humans. ST4 is common in rodents, and in some human populations (mainly Europe it seems), but otherwise extremely uncommon. And so on. This means that some subtypes may be difficult to establish in experimental animals. It also means that any pathology detected in the animal, may not be “reproducible” in another host, - maybe due to the fact that this host has adapted to this particular subtype or even strain. Blastocystis is common in a huge variety of animals, and different animals may have adapted do different subtypes. It is not unlikely that this is due to co-evolution, and therefore it may not turn out to be a big surprise if Blastocystis per se is not usually directly associated with disease. It may still be so, however, that for humans, some subtypes or strains may be associated with disease, preliminary data point in this direction.

3) Study design. Another issue is the use of appropriate controls – for example, experimental infection of animals with Blastocystis from cultures growing with bacteria need to have the appropriate controls - namely infection with the accompanying bacterial flora alone – before it can be concluded that Blastocystis is responsible for any effects seen. It is extremely difficult to axenise (i.e. make sterile) Blastocystis strains, so they will always be accompanied by some bacterial species. Hence, any effect noticed after challenge with a Blastocystis strain will be difficult to interpret, - is it due to Blastocystis or to accompanying bacterial strains? (If you want to see what Blastocystis look like in culture, go to my previous blog post here.)

So, results from scientific studies using animal experimental models should be interpreted cautiously. In vitro experimental models using enterocyte mono-layers for instance may constitute a more attractive alternative, but the problems of using xenic (i.e. unsterile) strains are evident also here. A great challenge ahead is the development of a standardised method for axenising (sterilising) strains… so far, such a method does not exist.

Our French colleagues recently published the genome of Blastocystis sp. ST7. Functional genomic analysis is key to understanding the extent to which Blastocystis is capable of exerting any direct pathological effect, and will assist us in studying the potential pathogenicity of Blastocystis in the absence of a suitable animal model. Indirect pathological effects may be more difficult to identify and probably require studies of the interaction between the host, the parasite and the rest of the gut microbiota (bacteria). Given our recent technological advances, I believe that a pathway to knowledge lies in the study of Blastocystis in an ecological context. I think that we should get an understanding of: 1) Who are colonized with Blastocystis, 2) From where do we get it, 3) For how long do we have the parasite, and do we establish symptoms in the very beginning, only to adapt to the presence of the parasite later on, 4) does Blastocystis require a particular flora to establish (and are there differences between subtypes (in humans and animals)), 5) could Blastocystis be seen as a proxy for a given gut microbiota (biomarker), and/or does Blastocystis select for a given microbiota phenotype (metatranscriptomic analysis of the intestinal flora accompanying Blastocystis might be useful to study how the bacteria “behave” (i.e. gene expression) in the presence/absence of Blastocystis), 6) are any Blastocystis-induced symptoms related to parasite abundance, etc.; this can be explored in rough detail by using real-time PCR, of which two have been published.

So, while animal models may not be immediately suitable in our quest to study Blastocystis pathogenicity, our “omics” methodologies and data analyses may sooner than we know help us answer many of the questions that we have been pondering for decades.

Having said that, I think that for instance a pig experimental model might be useful in terms of studying the effect of chemotherapeutic intervention. Obvious studies include those aiming to identify drugs capable of eradicating Blastocystis, but it could also be interesting to study the structure and function (gene expression profiling) of the accompanying microbiota before and after intervention.
Since pig feed often contains a range of antibiotics, it could be interesting to test whether pigs on diets +/- antibiotics differ in terms of Blastocystis colonisation... a recent PNAS paper demonstrates a shift in the structure and function of the microbiome in medicated pigs compared to pigs fed a diet void of antibiotics.

Further reading:

Chandramathi S, Suresh KG, Mahmood AA, & Kuppusamy UR (2010). Urinary hyaluronidase activity in rats infected with Blastocystis hominis--evidence for invasion? Parasitology research, 106 (6), 1459-63 PMID: 20358228

Elwakil HS, & Hewedi IH (2010). Pathogenic potential of Blastocystis hominis in laboratory mice. Parasitology research, 107 (3), 685-9 PMID: 20499092

Hussein EM, Hussein AM, Eida MM, & Atwa MM (2008). Pathophysiological variability of different genotypes of human Blastocystis hominis Egyptian isolates in experimentally infected rats. Parasitology research, 102 (5), 853-60 PMID: 18193282 

Iguchi A, Ebisu A, Nagata S, Saitou Y, Yoshikawa H, Iwatani S, & Kimata I (2007). Infectivity of different genotypes of human Blastocystis hominis isolates in chickens and rats. Parasitology international, 56 (2), 107-12 PMID: 17251054

Looft T, Johnson TA, Allen HK, Bayles DO, Alt DP, Stedtfeld RD, Sul WJ, Stedtfeld TM, Chai B, Cole JR, Hashsham SA, Tiedje JM, & Stanton TB (2012). In-feed antibiotic effects on the swine intestinal microbiome. Proceedings of the National Academy of Sciences of the United States of America, 109 (5), 1691-6 PMID: 22307632

Scanlan PD (2012). Blastocystis: past pitfalls and future perspectives. Trends in parasitology PMID: 22738855

Stensvold CR, Alfellani MA, Nørskov-Lauritsen S, Prip K, Victory EL, Maddox C, Nielsen HV, & Clark CG (2009). Subtype distribution of Blastocystis isolates from synanthropic and zoo animals and identification of a new subtype. International journal for parasitology, 39 (4), 473-9 PMID: 18755193

Stensvold CR (2012). Thinking Blastocystis out of the box. Trends in parasitology PMID: 22704911

Yan Y, Su S, Ye J, Lai X, Lai R, Liao H, Chen G, Zhang R, Hou Z, & Luo X (2007). Blastocystis sp. subtype 5: a possibly zoonotic genotype. Parasitology research, 101 (6), 1527-32 PMID: 17665214